DIFFERENT CARBOXYL-TERMINAL DOMAIN KINASE-ACTIVITIES ARE INDUCED BY HEAT-SHOCK AND ARSENITE - CHARACTERIZATION OF THEIR SUBSTRATE-SPECIFICITY, SEPARATION BY MONO-Q CHROMATOGRAPHY, AND COMPARISON WITH THE MITOGEN-ACTIVATED PROTEIN-KINASES

In response to heat-shock and chemical treatments, cells undergo profo und biochemical changes such as modifications in protein phosphorylati on in order to resist the new, unfavorable growth conditions. We have previously shown that in HeLa cells a protein kinase (HS-CTD kinase) a ctivity is induced rapidly after a heat or sodium arsenite shock. This kinase activity is able to phosphorylate a synthetic peptide composed of four repeats of the motif Ser-Pro Thr-Ser-Pro-Ser-Tyr, a motif hig hly repeated in the carboxyl-terminal domain (CTD) of the largest subu nit of eukaryotic RNA polymerase II. In this paper, we designed a new experimental procedure to characterize the substrate specificity of th is kinase activity. We show that HS-CTD kinase activity phosphorylates a consensus sequence (-P-X-S/T-P-) which is similar to the sequence p hosphorylated by extracellular regulated protein kinases (also called mitogen-activated protein kinases). However, there is a slight but rep roducible difference between these kinases in their use of serine or t hreonine as the phosphate acceptor. Mono Q chromatography allows the s eparation of five stress-induced CTD kinase activities, two of which c oelute with active mitogen-activated protein kinase forms revealed by Western blotting with anti ERK1-ERK2 antibodies. The other three CTD k inase activities induced after a stress are distinct from ERK1 and ERK 2 and have different enzymatic properties. The molecular nature of the se HS-CTD kinases and the physiological significance of their activati on during stress remain to be determined.